Biomedical subjects

Malcolm J Hawkesford

Publications and source records attributed to Malcolm J Hawkesford.

16 recordsLinked to original sources

PubMed2026

Seed-type vacuolar processing enzymes recognize the 619th asparagine residue to posttranslationally cleave the HMW-GS 1Dy10-m619SN allele.

High molecular weight glutenin subunits (HMW-GSs) are critical grain storage proteins in wheat, which govern its unique processing quality. A HMW-GS 1Dy10 allele variant (1Dy10-m619SN), carrying a serine-to-asparagine substitution at the 619th residue, undergoes partial posttranslational cleavage. This modification leads to improved cookie-making quality. However, the enzymes mediating this cleavage remain unknown. In this study, we identified vacuolar processing enzymes (VPEs) as candidates for 1Dy10-m619SN processing using TurboID-based proximity labeling and RNA-seq analysis. In vitro cleavage assays confirmed that VPEs catalyzed 1Dy10-m619SN cleavage. Phylogenic analysis revealed that there are two seed-type VPEs in wheat, TaVPEI and TaVPEII, with TaVPEI being further subdivided into TaVPEI-1, TaVPEI-2, and TaVPEI-3. Despite sharing conserved catalytic domains, these isoforms display distinct temporal expression patterns, with TaVPEI-1 expression showing the strongest correlation with the posttranslational cleavage of 1Dy10-m619SN. TaVPEI-1 protein is localized to the vacuole, the well-known deposition site for HMW-GSs. Overexpression of TaVPEI-1 in wheat enhances the 1Dy10-m619SN cleavage. Collectively, these findings demonstrate that the seed-type VPEs in wheat are responsible for the posttranslational cleavage of 1Dy10-m619SN, which provides new insights into the molecular basis of wheat's unique processing quality.

Triticum↗

PubMed2006

Dynamics of Aerenchyma distribution in the cortex of sulfate-deprived adventitious roots of maize.

BACKGROUND AND AIMS: Aerenchyma formation in maize adventitious roots is induced in nutrient solution by the deprivation of sulfate (S) under well-oxygenated conditions. The aim of this research was to examine the extent of aerenchyma formation in the cortex of sulfate-deprived adventitious roots along the root axis, in correlation with the presence of reactive oxygen species (ROS), calcium levels and pH of cortex cells and root lignification. METHODS: The morphometry of the second whorl of adventitious (W2) roots, subject to S-deprivation conditions throughout development, was recorded in terms of root length and lateral root length and distribution. W2 roots divided into sectors according to the mean length of lateral roots, and cross-sections of each were examined for aerenchyma. In-situ detection of alterations in ROS presence, calcium levels and pH were performed by means of fluorescence microscopy using H(2)DCF-DA, fluo-3AM and BCECF, respectively. Lignification was detected using the Wiesner test. KEY RESULTS: S-deprivation reduced shoot growth and enhanced root proliferation. Aerenchyma was found in the cortex of 77 % of the root length, particularly in the region of emerging or developing lateral roots. The basal and apical sectors had no aerenchyma and no aerenchyma connection was found with the shoot. S-deprivation resulted in alterations of ROS, calcium levels and pH in aerenchymatous sectors compared with the basal non-aerenchymatous region. Lignified epidermal layers were located at the basal and the proximal sectors. S-deprivation resulted in shorter lateral roots in the upper sectors and in a limited extension of the lignified layers towards the next lateral root carrying sector. CONCLUSIONS: Lateral root proliferation is accompanied by spatially localized induced cell death in the cortex of developing young maize adventitious roots during S-deprivation.

Calcium↗

PubMed2006

Biofortification of UK food crops with selenium.

Se is an essential element for animals. In man low dietary Se intakes are associated with health disorders including oxidative stress-related conditions, reduced fertility and immune functions and an increased risk of cancers. Although the reference nutrient intakes for adult females and males in the UK are 60 and 75 microg Se/d respectively, dietary Se intakes in the UK have declined from >60 microg Se/d in the 1970s to 35 microg Se/d in the 1990s, with a concomitant decline in human Se status. This decline in Se intake and status has been attributed primarily to the replacement of milling wheat having high levels of grain Se and grown on high-Se soils in North America with UK-sourced wheat having low levels of grain Se and grown on low-Se soils. An immediate solution to low dietary Se intake and status is to enrich UK-grown food crops using Se fertilisers (agronomic biofortification). Such a strategy has been adopted with success in Finland. It may also be possible to enrich food crops in the longer term by selecting or breeding crop varieties with enhanced Se-accumulation characteristics (genetic biofortification). The present paper will review the potential for biofortification of UK food crops with Se.

Crops, Agricultural↗

PubMed2006

Managing sulphur metabolism in plants.

Resolution and analysis of genes encoding components of the pathways of primary sulphur assimilation have provided the potential to elucidate how sulphur is managed by plants. Individual roles for members of gene families and regulatory mechanisms operating at gene, cellular and whole plant levels have been recognized. Sulphur is taken up and transported around the plant principally as sulphate, catalysed for the most part by a single gene family of highly regulated transporters. Additional regulation occurs in the pathway of reduction of sulphate to sulphide and its incorporation into cysteine, which occurs principally within the plastid. Cellular and whole-plant regulation of uptake, and the assimilatory pathway attempt to balance supply with demand for growth and include mechanisms for re-mobilization and redistribution of sulphur. Furthermore, optimization of sulphur assimilation requires coordination with carbon and nitrogen pathways, and multiple processes have been proposed to contribute to this balance. Present studies on cis and trans elements are focusing on transcriptional regulation, but this regulation still needs to be linked to apparent metabolite sensing. Whilst the components of the assimilatory pathways have been resolved after many years of controversy, uncertainties remain concerning roles of individual genes in gene families, their sub-cellular localization and their significance in balancing sulphur flux to sulphur demand of the plant for growth under variable environmental conditions.

Anion Transport Proteins↗

PubMed2005

Markedly different gene expression in wheat grown with organic or inorganic fertilizer.

Nitrogen is the major determinant of crop yield and quality and the precise management of nitrogen fertilizer is an important issue for farmers and environmentalists. Despite this, little is known at the level of gene expression about the response of field crops to different amounts and forms of nitrogen fertilizer. Here we use expressed sequence tag (EST)-based wheat microarrays in combination with the oldest continuously running agricultural experiment in the world to show that gene expression is significantly influenced by the amount and form of nitrogenous fertilizer. In the Broadbalk winter wheat experiment at Rothamsted in the United Kingdom and at three other diverse test sites, we show that specific genes have surprisingly different expression levels in the grain endosperm when nitrogen is supplied either in an organic or an inorganic form. Many of the genes showing differential expression are known to participate in nitrogen metabolism and storage protein synthesis. However, others are of unknown function and therefore represent new leads for future investigation. Our observations show that specific gene expression is diagnostic for use of organic sources of nitrogen fertilizer and may therefore have useful applications in defining the differences between organically and conventionally grown wheat. [The sequences reported in this paper have been deposited in the GenBank database (accession nos. AL 208216-AL 831324).]

Base Sequence↗

PubMed2005

Using a suppression subtractive library-based approach to identify tobacco genes regulated in response to short-term sulphur deficit.

Monitoring expression at the transcriptional level is an essential first step for the functional analysis of plant genes. Genes encoding proteins directly involved in sulphur metabolism constitute only a small fraction of all the genes affected by sulphur deficiency stress. Transcriptional responses to various periods of sulphur deprivation have been extensively studied in Arabidopsis thaliana; however, no corresponding data are available for Solanaceae sp. To address this problem, a subtractive library-based approach to search for tobacco genes regulated by a short-term sulphur starvation has been adopted. In this work, 38 genes were identified, of which 22 were regulated positively and 16 were regulated negatively. The transcript levels of the representative genes were monitored in four parts of the plants (mature and immature leaves, stems, and roots), which exhibited differential sulphur deficiency. Interestingly, some genes exhibit different regulation of expression in different parts of the plants. Database analysis allowed assignment of the potential function for many of the identified genes; however, the functions of a small number of genes strongly regulated by sulphur starvation remain unknown. The genes were grouped into nine functional categories, each including both up- and down-regulated genes. The possible links between the identified regulated genes and sulphur metabolism are considered, and compared where possible with expression patterns in Arabidopsis thaliana. Although no obvious regulatory genes were identified, the genes encoding proteins of unknown function remain as potential components of the regulatory processes.

Down-Regulation↗

PubMed2005

Systems rebalancing of metabolism in response to sulfur deprivation, as revealed by metabolome analysis of Arabidopsis plants.

Sulfur is an essential macro-element in plant and animal nutrition. Plants assimilate inorganic sulfate into two sulfur-containing amino acids, cysteine and methionine. Low supply of sulfate leads to decreased sulfur pools within plant tissues. As sulfur-related metabolites represent an integral part of plant metabolism with multiple interactions, sulfur deficiency stress induces a number of adaptive responses, which must be coordinated. To reveal the coordinating network of adaptations to sulfur deficiency, metabolite profiling of Arabidopsis has been undertaken. Gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry techniques revealed the response patterns of 6,023 peaks of nonredundant ion traces and relative concentration levels of 134 nonredundant compounds of known chemical structure. Here, we provide a catalogue of the detected metabolic changes and reconstruct the coordinating network of their mutual influences. The observed decrease in biomass, as well as in levels of proteins, chlorophylls, and total RNA, gives evidence for a general reduction of metabolic activity under conditions of depleted sulfur supply. This is achieved by a systemic adjustment of metabolism involving the major metabolic pathways. Sulfur/carbon/nitrogen are partitioned by accumulation of metabolites along the pathway O-acetylserine to serine to glycine, and are further channeled together with the nitrogen-rich compound glutamine into allantoin. Mutual influences between sulfur assimilation, nitrogen imbalance, lipid breakdown, purine metabolism, and enhanced photorespiration associated with sulfur-deficiency stress are revealed in this study. These responses may be assembled into a global scheme of metabolic regulation induced by sulfur nutritional stress, which optimizes resources for seed production.

Amino Acids↗

PubMed2005

O-acetylserine and the regulation of expression of genes encoding components for sulfate uptake and assimilation in potato.

cDNAs encoding a high-affinity sulfate transporter and an adenosine 5'-phosphosulfate reductase from potato (Solanum tuberosum L. cv Desiree) have been cloned and used to examine the hypothesis that sulfate uptake and assimilation is transcriptionally regulated and that this is mediated via intracellular O-acetylserine (OAS) pools. Gas chromotography coupled to mass spectrometry was used to quantify OAS and its derivative, N-acetylserine. Treatment with external OAS increased sulfate transporter and adenosine 5'-phosphosulfate reductase gene expression consistent with a model of transcriptional induction by OAS. To investigate this further, the Escherichia coli gene cysE (serine acetyltransferase EC 2.3.1.30), which synthesizes OAS, has been expressed in potato to modify internal metabolite pools. Transgenic lines, with increased cysteine and glutathione pools, particularly in the leaves, had increased sulfate transporter expression in the roots. However, the small increases in the OAS pools were not supportive of the hypothesis that this molecule is the signal of sulfur (S) nutritional status. In addition, although during S starvation the content of S-containing compounds decreased (consistent with derepression as a mechanism of regulation), OAS pools increased only following extended starvation, probably as a consequence of the S starvation. Taken together, expression of these genes may be induced by a demand-driven model, via a signal from the shoots, which is not OAS. Rather, the signal may be the depletion of intermediates of the sulfate assimilation pathway, such as sulfide, in the roots. Finally, sulfate transporter activity did not increase in parallel with transcript and protein abundance, indicating additional posttranslational regulatory mechanisms.

Acetyltransferases↗

PubMed2004

Regulation of sulfate uptake and expression of sulfate transporter genes in Brassica oleracea as affected by atmospheric H(2)S and pedospheric sulfate nutrition.

Demand-driven signaling will contribute to regulation of sulfur acquisition and distribution within the plant. To investigate the regulatory mechanisms pedospheric sulfate and atmospheric H(2)S supply were manipulated in Brassica oleracea. Sulfate deprivation of B. oleracea seedlings induced a rapid increase of the sulfate uptake capacity by the roots, accompanied by an increased expression of genes encoding specific sulfate transporters in roots and other plant parts. More prolonged sulfate deprivation resulted in an altered shoot-root partitioning of biomass in favor of the root. B. oleracea was able to utilize atmospheric H(2)S as S-source; however, root proliferation and increased sulfate transporter expression occurred as in S-deficient plants. It was evident that in B. oleracea there was a poor shoot to root signaling for the regulation of sulfate uptake and expression of the sulfate transporters. cDNAs corresponding to 12 different sulfate transporter genes representing the complete gene family were isolated from Brassica napus and B. oleracea species. The sequence analysis classified the Brassica sulfate transporter genes into four different groups. The expression of the different sulfate transporters showed a complex pattern of tissue specificity and regulation by sulfur nutritional status. The sulfate transporter genes of Groups 1, 2, and 4 were induced or up-regulated under sulfate deprivation, although the expression of Group 3 sulfate transporters was not affected by the sulfate status. The significance of sulfate, thiols, and O-acetylserine as possible signal compounds in the regulation of the sulfate uptake and expression of the transporter genes is evaluated.

Atmosphere↗

PubMed2004

Vacuolar sulfate transporters are essential determinants controlling internal distribution of sulfate in Arabidopsis.

Uptake of external sulfate from the environment and use of internal vacuolar sulfate pools are two important aspects of the acquisition of sulfur for metabolism. In this study, we demonstrated that the vacuolar SULTR4-type sulfate transporter facilitates the efflux of sulfate from the vacuoles and plays critical roles in optimizing the internal distribution of sulfate in Arabidopsis thaliana. SULTR4;1-green fluorescent protein (GFP) and SULTR4;2-GFP fusion proteins were expressed under the control of their own promoters in transgenic Arabidopsis. The fusion proteins were accumulated specifically in the tonoplast membranes and were localized predominantly in the pericycle and xylem parenchyma cells of roots and hypocotyls. In roots, SULTR4;1 was constantly accumulated regardless of the changes of sulfur conditions, whereas SULTR4;2 became abundant by sulfur limitation. In shoots, both transporters were accumulated by sulfur limitation. Vacuoles isolated from callus of the sultr4;1 sultr4;2 double knockout showed excess accumulation of sulfate, which was substantially decreased by overexpression of SULTR4;1-GFP. In seedlings, the supplied [(35)S]sulfate was retained in the root tissue of the sultr4;1 sultr4;2 double knockout mutant. Comparison of the double and single knockouts suggested that SULTR4;1 plays a major role and SULTR4;2 has a supplementary function. Overexpression of SULTR4;1-GFP significantly decreased accumulation of [(35)S]sulfate in the root tissue, complementing the phenotype of the double mutant. These results suggested that SULTR4-type transporters, particularly SULTR4;1, actively mediate the efflux of sulfate from the vacuole lumen into the cytoplasm and influence the capacity for vacuolar storage of sulfate in the root tissue. The efflux function will promote rapid turnover of sulfate from the vacuoles particularly in the vasculature under conditions of low-sulfur supply, which will optimize the symplastic (cytoplasmic) flux of sulfate channeled toward the xylem vessels.

Arabidopsis↗

PubMed2004

Plant sulphate transporters: co-ordination of uptake, intracellular and long-distance transport.

Proton/sulphate co-transport in the plasma membrane of root cells is the first step for the uptake of sulphate from the environment by plants. Further intracellular, cell-to-cell and long-distance transport must fulfil the requirements for sulphate assimilation and source/sink demands within the plant. A gene family of sulphate transporters, which may be subdivided into five groups, has been identified with examples from many different plant species. For at least two groups, proton/sulphate co-transport activity has been confirmed. It appears that each group represents sulphate transporters with distinct kinetic properties, patterns of expression, and cell/tissue specificity related to specific roles in the uptake and allocation of sulphate. High-affinity sulphate uptake and low-affinity vascular transport, as well as vacuolar efflux, are controlled by the nutritional status of the plant. Most notably there is an apparent increase in capacity for cellular sulphate uptake and vacuolar efflux when sulphur supply is limiting. Within the groups, the individual sulphate transporters may be further subdivided by differences in temporal, cellular and tissue expression. Many of the transporters are regulated by the nutritional status of the individual tissues, to optimize sulphate movement within and between sink and source organs.

Biological Transport↗

PubMed2004

Phylogeny and expression of paralogous and orthologous sulphate transporter genes in diploid and hexaploid wheats.

Twelve genes encoding two closely related subtypes (ST1.1a and ST1.1b) of a sulphate transporter have been identified in the diploid wheats Aegilops tauschii, Triticum urartu, and Aegilops speltoides, as well as the hexaploid Triticum aestivum. Based on phylogenetic comparisons with other plant sulphate transporters, the ST1.1a and 1.1b subtypes aligned with group 1 of the plant sulphate transporter gene family. The exon-intron structure was conserved within the ST1.1a or ST1.1b genes; however, substantial variability in intron sequences existed between the two types. The high overall sequence similarity indicated that ST1.1b represented a duplication of the ST1.1a gene, which must have occurred before the evolution of the ancestral diploid wheat progenitor. In contrast with the close relationship of the T. urartu and Ae. tauschii sequences to the corresponding A and D genome sequences of T. aestivum, the divergence between the Ae. speltoides sequences and the B genome sequences suggested that the B genome ST1.1a gene has been modified by recombination. Transcript analysis revealed predominant expression of the ST1.1a type and an influence of sulphur availability on the level of expression.

Base Sequence↗

PubMed2003

Cloning of two contrasting high-affinity sulfate transporters from tomato induced by low sulfate and infection by the vascular pathogen Verticillium dahliae.

Two cDNAs, LeST1-1 (AF347613) and LeST1-2 (AF347614), encoding sulfate transporters have been cloned from tomato (Lycopersicon esculentum Mill.) by reverse transcription-polymerase chain reaction and their expression characterised. Sharing 76% identity at the amino acid level, the transporters are phylogenetically associated with the Group-1, high-affinity plant sulfate transporters. Both were shown to have high affinity for sulfate by uptake kinetic analysis using a yeast (Saccharomyces cerevisiae) sulfate-transporter mutant. Km values of 11.5 microM and 9.8 microM were calculated for LeST1-1 and LeST1-2, respectively, the same order of magnitude as those previously reported for several other Group-1 high-affinity sulfate transporters. In situ hybridisation to S-deficient tomato roots showed LeST1-1 to be expressed in the epidermis and pericycle, whereas LeST1-2 expression was located to the epidermis only. Northern analysis shows that the mRNA abundances of both LeST1-1 and LeST1-2 are upregulated in the root in response to sulfate deprivation. LeST1-1 is specifically expressed in root tissue, a characteristic of Group-1 sulfate transporters. LeST1-2, however, was also detected in tomato leaves and stems and is upregulated and expressed to a similar extent in these tissues under conditions of sulfate deprivation. Induction of LeST1-2 expression was also observed in the vascular tissues of a resistant line of tomato infected with the vascular wilt pathogen Verticillium dahliae.

Base Sequence↗

PubMed2002

Modulation of cyanoalanine synthase and O-acetylserine (thiol) lyases A and B activity by beta-substituted alanyl and anion inhibitors.

The reaction mechanisms of three enzymes belonging to a single gene family are compared: a cyanoalanine synthase and two isoforms of O-acetylserine (thiol) lyase (O-ASTL) isolated from spinach (Spinacea oleracea L. cv. Medina). O-ASTL represents a major regulatory point in the S-assimilatory pathway, and the related cyanoalanine synthase, which is specific to the mitochondrial compartment, has evolved an independent function of cyanide detoxification. All three enzymes catalysed both the cysteine synthesis and cyanoalanine synthesis reactions although with different efficiencies, and which may be explained by a single amino acid substitution in the substrate-binding pocket of the enzyme. Substituted alanine and nucleophillic inhibitors caused predominantly non-competitive inhibition, indicating binding to both E- and F-forms of the enzyme in a bi-bi ping-pong kinetic model. Michaelis-Menten kinetics were observed when the alanyl substrate was varied in the presence and absence of inhibitors. The use of alanyl inhibitors has shown that the alanyl half-cycle of both the cysteine synthesis and cyanoalanine synthesis reactions of cyanoalanine synthase and O-acetylserine (thiol) lyases are similar. This is in contrast to the results observed with nucleophillic inhibitors, which have shown that the mechanisms of anion binding and processing differ between cyanoalanine synthase and O-ASTLs.

Alanine↗

PubMed2002

Influence of iron status on cadmium and zinc uptake by different ecotypes of the hyperaccumulator Thlaspi caerulescens.

We have previously identified an ecotype of the hyperaccumulator Thlaspi caerulescens (Ganges), which is far superior to other ecotypes (including Prayon) in Cd uptake. In this study, we investigated the effect of Fe status on the uptake of Cd and Zn in the Ganges and Prayon ecotypes, and the kinetics of Cd and Zn influx using radioisotopes. Furthermore, the T. caerulescens ZIP (Zn-regulated transporter/Fe-regulated transporter-like protein) genes TcZNT1-G and TcIRT1-G were cloned from the Ganges ecotype and their expression under Fe-sufficient and -deficient conditions was analyzed. Both short- and long-term studies revealed that Cd uptake was significantly enhanced by Fe deficiency only in the Ganges ecotype. The concentration-dependent kinetics of Cd influx showed that the V(max) of Cd was 3 times greater in Fe-deficient Ganges plants compared with Fe-sufficient plants. In Prayon, Fe deficiency did not induce a significant increase in V(max) for Cd. Zn uptake was not influenced by the Fe status of the plants in either of the ecotypes. These results are in agreement with the gene expression study. The abundance of ZNT1-G mRNA was similar between the Fe treatments and between the two ecotypes. In contrast, abundance of the TcIRT1-G mRNA was greatly increased only in Ganges root tissue under Fe-deficient conditions. The present results indicate that the stimulatory effect of Fe deficiency on Cd uptake in Ganges may be related to an up-regulation in the expression of genes encoding for Fe(2+) uptake, possibly TcIRT1-G.

Arabidopsis Proteins↗

PubMed2002

Elemental sulfur and thiol accumulation in tomato and defense against a fungal vascular pathogen.

The occurrence of fungicidal, elemental S is well documented in certain specialized prokaryotes, but has rarely been detected in eukaryotes. Elemental S was first identified in this laboratory as a novel phytoalexin in the xylem of resistant genotypes of Theobroma cacao, after infection by the vascular, fungal pathogen Verticillium dahliae. In the current work, this phenomenon is demonstrated in a resistant line of tomato, Lycopersicon esculentum, in response to V. dahliae. A novel gas chromatography-mass spectroscopy method using isotope dilution analysis with 34S internal standard was developed to identify unambiguously and quantify 32S in samples of excised xylem. Accumulation of S in vascular tissue was more rapid and much greater in the disease-resistant than in the disease-susceptible line. Levels of S detected in the resistant variety (approximately 10 microg g-1 fresh weight excised xylem) were fungitoxic to V. dahliae (spore germination was inhibited >90% at approximately 3 microg mL-1). Scanning electron microscopy-energy dispersive x-ray microanalysis confirmed accumulation of S in vascular but not in pith cells and in greater amounts and frequency in the Verticillium spp.-resistant genotype. More intensive localizations of S were occasionally detected in xylem parenchyma cells, vessel walls, vascular gels, and tyloses, structures in potential contact with and linked with defense to V. dahliae. Transient increases in concentrations of sulfate, glutathione, and Cys of vascular tissues from resistant but not susceptible lines after infection may indicate a perturbation of S metabolism induced by elemental S formation; this is discussed in terms of possible S biogenesis.

Cacao↗